This SBIR project is focused on developing a novel 3D culture system that incorporates a unique HuBiogel scaffold, supports growth/expansion of cancer stem cells (CSCs) and allows precise cellular and molecular analysis. At present, spheroid cultures and synthetic or animal-derived matrices are commonly used by cancer researchers. However, these do not fully mimic heterotypic microenvironment and fail to retain CSCs viability or stem-like properties. VBI has developed a 3D HuBiogel system that supports multicellular growth and maintains phenotypic status of normal and tumor cells. Pilot studies demonstrate tumor-derived CSCs can be encapsulated in HuBiogel+MSC matrix spheres with high growth/viability, the essential criteria for functional analysis. Phase I objectives are: 1) Develop a reliable 3D culture system for CSCs using an integrated HuBiogel-microsphere-bioreactor cultivation approach; 2) Evaluate expansion and biomarker profiles of CSCs in new 3D HuBiogel culture system under physiologic microenvironments (hypoxia, low glucose); and 3) Validate CSCs exhibit/retain tumorigenic potential using functional read outs (colony assay and tumorgraft model). Established human brain tumor lines (GBM) will be used for optimization of 3D culture metrics. Results will be compared to standard cell spheroid controls. In extended phase, adaptability of this fully-human CSCs cultivation system for biologic, proteomic and genomic analyses will be demonstrated. We are confident of successfully developing a marketable 3D culture/analysis system and products useful for growing CSCs for basic and translational research.